DE69533623T2 - METHOD FOR SUPPORTING DIAGNOSIS OF ALZHEIMER'S DISEASE BY MEASUREMENT OF AMYLOID BETA PEPTIDES (X MORE THAN OR OR 41) AND TAU - Google Patents

Abstract

Diagnosis and monitoring of Alzheimer's disease (AD) is aided by: (a) measuring the amount of at least one soluble Abeta (x->= 41) peptide (I) in a test sample; (b) comparing the result with a predetermined amount of the same peptide; and (c) assessing patient status from the difference.

Description

Background of the invention

1. Field of the invention

The The present invention generally relates to methods of diagnosis and monitoring Alzheimer's disease. More specifically, the present invention relates Invention measuring the amount of tau protein and / or the amount of β-amyloid Peptide (x-≥ 41) in fluid samples of patients, as well as the use of these quantities as a diagnostic Indicator.

The Alzheimer's disease (Alzheimer's disease, AD) is characterized by a degenerative brain disease clinically by the progressive loss of memory, the ability to recognize, of logical reasoning, judgmental as well as emotional Stability, what gradually to severe mental damage and ultimately leads to death. AD is a very common one Cause for progressive mental dysfunction (dementia) in people progressed Age and is believed to be the fourth most common medical Cause for Representing death in the United States. AD was in all races and observes ethnic groups worldwide and constitutes one of the meant current and future public health problems This disease - so estimates man - concerns currently about two to three million individuals in the United States. AD is currently incurable. No treatment is currently known which effectively prevents AD or restricts its symptoms.

The Brains of individuals with AD show characteristic lesions, called senile plaques and neurofibrillary tangles. A variety of these lesions are generally found in several regions of the human Brain, important for the memory and the detection function, for Patients with AD. A smaller number of these lesions can be found in one limited anatomical distribution often in the brains of the Age-advanced individuals who have no clinical AD signs exhibit. Characterize senile plaques and amyloid angiopathy also the brains of individuals over half of a certain age with trisomy 21 (Down syndrome) and heriditary cerebral hemorrhage with Dutch-type amyloidosis (HCHWA-D, Hereditary Cerebral Hemorrhage with amyloidosis of the Dutch type). Currently requires a definite Diagnosis of Alzheimer's usually the observation of the above lesions in the brain tissue of the Patients who died with the disease or - more rarely Cases - in small Biopsy samples taken from brain tissue during an interventional neurosurgical Procedure. The principal chemical component of senile plaques and the vascular amyloid deposits (amyloid angiopathy) that are characteristic for AD and the other illnesses mentioned above is about 4.2 Kilodalton (kD) large Protein of about 39-43 Amino acids, called the amyloid β-peptide (Aβ) or sometimes βAP, AβP or β / A4. The first purification Aβ as well the first report of a partial amino acid sequence is from Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120: 885-890. The Isolation procedure and the sequence data for the first 28 amino acids become in U.S. Patent No. 4,666,829. Forms of Aβ with amino acids of less than 40 were first reported by Kang et al. (1987) Nature 325: 733-736.

raw et al. (1993) Proc. Natl. Acad. Sci. USA 90: 10836-840 showed that Aβ (1-42) of the Main component in neuritic (= nerve) plaques (90%) is and although with significant amounts of isomerized and racemized Aspartyl. The authors also showed that Aβ (17-42) also in diffuse plaques (70%), whereas Aβ (1-40) predominates Main component in meningo-vascular plaques is comprehensive 60% of the total Aβ and in parenchymal vessels deposits from Aβ (1-42) 75% represent the entire Aβ. Iwatsubo et al. (1994) Neuron 13: 45-53 showed that Aβ42 (43) was positive senile plaques the main Species in sporadic AD brains is.

molecular biology and have protein-chemical analyzes done during the last years shown that Aβ small fragment of a much larger precursor protein which is called the β-amyloid precursor protein (Β-amyloid precursor protein, APP), which is normally derived from cells in many tissues of different animals, including humans, is produced. Knowledge of the structure of the gene, which APP coded, has demonstrated that Aβ as a peptide fragment appears from the carboxyl terminus of APP from previously unknown enzymes (protease) cut off becomes. The precise biochemical mechanism by which the Aβ fragment is cut off from the APP and subsequently as amyloid plaques in the cerebral tissues and in the walls cerebral and meningeal blood vessels is deposited currently unknown.

Various Lines of evidence indicate that the progression of cerebral Deposition of Aβ one fundamental role in the pathogenesis of AD plays and cognitive Symptoms precedes years or decades (for a review, see Selkoe (1994) J. Neuropath. and Exp. Neurol. 53: 438-447 and Selkoe (1991) Neuron 6: 487). The most prominent, most important chain of evidence is the discovery from 1991 that missense DNA mutations at amino acid 717 the 770 amino acid isoform APP in affected members, but not in non-affected Members of several families with a genetically determined (familial) form can be found by AD (Goate et al., (1991) Nature 349: 704-706; Chartier Harlan et al. (1991) Nature 353: 844-846; and Murrell et al. (1991) Science 254: 97--99). Suzuki et al. (1994) Science 264: 1336-1340 showed that in individuals with the 717 mutation a higher percentage of Aβ (1-42) as at Aβ (1-40) occurs.

In addition, a double mutation altering lysine ⁵⁹⁵ -methionine ⁵⁹⁶ to asparagine ⁵⁹⁵ -leucine ⁵⁹⁶ (based on the 695 isoform) found in Swedish families was reported in 1992 (Mullan et al. (1992) Nature Genet 1: 345 -347), and this is called the Swedish variant. Genetic linkage analyzes have demonstrated that these mutations, as well as certain other mutations of the APP gene, are the specific molecular cause of AD in the affected members of such families. In addition, a mutation of amino acid 693 of the 770 amino acid isoform of APP was identified as the cause of Aβ deposition disease HCHWA-D, and alteration of alanine to glycine at amino acid 692 appears to produce a phenotype that resembles AD in some patients, however HCHWA-D in others. The discovery of these and other mutations in APP in genetically-based cases of AD suggests that alteration of APP and subsequent deposition of the Aβ fragment may cause AD.

Neurofibrillary tangles consist primarily of the microtubulin protein, tau. Z. S. Khachaturian (1985) Arch. Neurol. 42: 1097-1105. Recent studies have shown that dew increases in the CSF of patients with Alzheimer's disease. M. Vandermeeren et al. (1993) J. Neurochem. 61: 1828-1834.

In spite of of progress, in understanding the underlying mechanisms of AD continue the need To develop methods of use in the diagnosis of these diseases. While the degree of dew provides some help in the diagnosis of Alzheimer's (M. Vandermeeren et al., supra) would be more markers and more specific Helpful. It would be furthermore, desirable method to provide for use in the diagnosis of Aβ-related conditions, the diagnosis being based, at least in part, on the detection of Aβ and Fragments in fluid samples based on patients. Specific assays for Aβ detection should be able his, Aβ and related fragments in fluid samples in very low concentrations, as well as between Aβ and others of APP that can be present in a sample.

2. Description of the state of the technique

Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120: 885-890 and U.S. Patent No. 4,666,829 are discussed above. The '829 patent specifies the use of an antibody against the 28-amino acid Aβ fragment close to the "Alzheimer's amyloid polypeptide" in a sample of a Diagnose patients for detection and Alzheimer's. None Data demonstrating the detection or diagnosis will become presented.

Various biochemical electron microscopic and immunochemical studies have reported that Aβ is highly insoluble in physiological solutions at normal pH. See, for example, Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 122: 1131-1135; Masters et al. (1985) Proc. Natl. Acad. Sci. USA 82: 4245-4249; Selkoe et al. (1986) J. Neurochem. 46: 1820-1834; Joachim et al. (1988) Brain Research 474: 100-111; Hilbich et al. (1991) J. Mol. Biol. 218: 149-163; Barrow and Zagorski (1991) Science 253: 179-182; and Burdick et al. (1992) J. Biol. Chem. 267: 546-554. Furthermore, this insolubility was predicted by and is consistent with the amino acid sequence Aβ, which includes a piece of hydrophobic amino acids that constitutes part of the region anchoring the parent protein (APP) in the lipid membranes of cells. Hydrophobic, lipid-anchoring proteins, such. As Aβ, are thought to remain associated with cellular membranes or membrane fragments and therefore not present in physiologically extracellular fluids. The above-mentioned studies and many others have reported the insolubility in physiological solution of native Aβ purified from AD brain amyloid deposits or from synthetic peptides containing the Aβ sequence. Extraction of Aβ from cerebral amyloid deposits and their subsequent solubilization has required the use of non-physiological solvents and denaturing agents. Physiological, buffered saline solutions containing the ex trapping tracelular fluids of human tissue have not been uniform enough to solubilize Aβ.

Further Attempts to detect APP or fragments thereof in plasma or in CSF, were also undertaken. A big secreted fragment from APP that does not have an intact Aβ region contains was found in human cerebrospinal fluid. (Palmert et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6338-6342; Weidemann et al. (1989) Cell 57: 115-126; Henriksson et al. (1991) J. Neurochem. 56: 1037-1042; Palmert et al. (1990) Neurology 40: 1028-1034; and Seubert et al. (1993) Nature 361: 260-263) as well as in plasma (Podlisny et al. (1990) Biochem. Biophys. Res. Commun. 167: 1094-1101). The detection of fragments of the carboxy-terminal portion of APP in Plasma has also been reported (Rumble et al., (1989) N. Engl. J. Ned. 320: 1446-1452), as well as the failure of the proof of these fragments (Schlossmacher et al. (1992) Neurobiol. Aging 13: 421-434).

In spite of the obvious insolubility of native and synthetic Aβ speculates that the Aβ is under circumstances in body fluids occurs, such. Cerebrospinal fluid (cerebrospinal fluid, CSF) or in plasma (Wong et al., (1984) Proc. Natl. Acad Sci., USA 92: 8729-8732; Selkoe (1986) Neurobiol. Aging 7: 425-432; Pardridge et al. (1987) Biochem. Biophys. Res. Commun. 145: 241-248; Joachim et al. (1989) Nature 341: 226-230; Selkoe et al. (1989) Neurobiol. Aging 10: 387-395).

From several trials, Aβ in CSF and plasma measurements were reported by both radioimmunoassay procedures (WO90 / 12870, published November 1, 1990) as well as sandwich ELISAs (Wisniewski in Alzheimer's disease, Ed. Becker and Giacobini, Taylor and Francas, N.Y. P. 206, 1990; Kim and Wisniewski in Techniques in Diagnostic Pathology, ed. Bullock et al., Academic Press, Boston, p. 106; and WO90 / 12871 on November 1, 1990). While These reports show very low levels of Aβ immunoreactivity in body fluids have been detected, no attempts of direct purification and further characterization of this immunoreactivity as well the determination of whether it represented Aβ, and the effort got employed. The possibility the Aβ production Cultured cells were never considered, nor ever shown.

Retrospective was the inability Aβ easy in body fluids evidence, probably for the presence of amyloid precursor fragments attributed, with overlapping Regions or fragments of Aβ, which obscured the measurements as well as the nonexistence of antibodies that Completely specific for intact Aβ. This is probably because the antibodies used by both Groups would cross-react with other APP fragments which Contain parts of Aβ, from which are known to be present in CSF, creating an interaction would be achieved with the measurement if anything one of intact Aβ succeeds. These difficulties have been overcome by the use of monoclonal antibodies, specific for an epitope in the central junction region of intact Aβ (Seubert et al. (1992) Nature 359: 325-327).

Seubert et al. (1992) Nature 359: 325-327 and Shoji et al. Science (1992) 258: 126-129 presented the first biochemical Proof for the presence of discrete Aβ in body fluids to disposal. Vigo-Pelfrey et al. (1993) J. Neurochem. 61: 1965-1968 reported from the identification of many Aβ species in cerebrospinal Liquid.

Summary of the invention

The The present invention provides methods useful for aiding in diagnosis as well as monitoring of Aβ-related Conditions in patients available, with the procedures themselves on the specific detection of fluid samples in patients of one or more soluble Aβ or soluble Aβ fragments leave, the amino acid residues have above the number 40 in their carboxy-terminus. These peptides are referred to as "Aβ (x-≥41)" (Aβ from the amino acid No "x" to an amino acid greater or equal to the amino acid No. 41). In one embodiment belong the measured peptides to the class of Aβ (x-≥41) which contain at least amino acids 13-41.

For the diagnosis and monitoring of Aβ-related conditions, the amount of the above-mentioned peptides in a fluid sample of a patient, especially in the cerebrospinal fluid (CSF), is measured and compared to a predetermined value, e.g. An indicator value (in the case of diagnosis) or an earlier patient value (in the case of monitoring). In the case of diagnosis, the measured values of Aβ (x-≥41) that are above the indicator value are considered strong indications that the patient is not suffering from AD or other Aβ-related conditions. However, this information can also be considered together with other factors in making a definitive diagnosis. Measured values of Aβ (x-≥41) that are at or below the indicator value are considered a positive indication that the patient has AD or other Aβ-related relationships. The low Aβ (x-≥41) status of the tested individual will not usually be considered diagnostic for an Aβ-related condition per se, but will be considered along with other accepted clinical symptoms of Aβ-related conditions in making the diagnosis. In cerebrospinal fluid, an indicator value of about 0.5 ng / ml is significant.

In In a specific aspect, the present invention provides specific ones Binding assays ready for the Detection of soluble Aβ (x-≥ 41) in fluid samples are significant, and those in for Patient diagnostic and monitoring Method, as just described, can be used.

specific Binding assays according to the present Invention employed two binding substances specific for different Epitopes or specific sites on the Aβ (x-≥41) molecule. An epitope or a Job is usually located not on other fragments or degradation products of the amyloid β-precursor protein (APP) to avoid cross-reactions with these fragments. Especially important are antibodies, which recognize a junction region within Aβ, the junction region around the site of the normal proteolytic interface of APP between residues Lys16 and Leu17 (Esch et al. (1990) Science 248: 492-495 and Anderson et al. (1991) Neuro. Science Lett. 128: 126-128), and typically by the amino acid residues 13 to 26 is stretched. The other epitope or the other place contain at least one amino acid via amino acid no. 40 from Aβ, which is essential for the recognition is, but not cross-reacted with Aβ or Aβ fragments, their carboxy-terminal amino acid No. 40 or lower. Exemplary specific binding assays shut down Two-site (sandwich) assays in which the capturing antibody is specific for the junction region of Aβ is, as previously described, and a second detectable antibody specific for a Epitope or a site containing at least one Aβ amino acid over the No. 40 is out. In particular, the second antibody may be Immunization with a hapten containing Aβ amino acids 33-42.

These The invention also provides methods for assisting in diagnosis or monitoring of Alzheimer's disease in a patient ready to take measurements from both Aβ (x-≥41) and the Microtubule protein include dew. The procedures include Measurement of the amount of one or more soluble Aβ (x-≥41) in a patient's sample one; the comparison of the measured quantity with a predetermined one Amount of soluble Aβ (x-≥41); the Measuring the amount of tau in the sample of a patient; the comparison the measured amount with a predetermined amount of said Dew; as well as evaluating the status of the patient based on a difference between the measured and predetermined values of Aβ (x-≥41) and Tau. Again, a predetermined amount may be an indicator value or a previous patient value be. A measured amount at or below the Aβ (x-≥41) indicator value and at or above the dew indicator value provides a positive Note in the diagnosis of Alzheimer's disease, where a measured Amount above the Aβ (x-≥41) indicator value and below the tau indicator value, a negative indication of Diagnosis of Alzheimer's Disease provides. indicator values in the CSF of about 0.5 ng / ml for Aβ (x-≥41) and about 0.3 ng / ml for dew are significant.

• a) einen ungelabelten Antikörper, welcher an die Verbindungsregion von Aβ bindet;
• b) einen nachweisbar gelabelten Antikörper, welcher ein Epitop enthaltend eine Aminosäure oberhalb Nr. 40 in Aβ bindet;
• c) einen ungelabelten Antikörper, welcher an Tau bindet; und
• d) einen nachweisbar gelabelten Antikörper, der an Tau bindet.

This invention also provides kits for aiding in the diagnosis of Alzheimer's disease. The kits include a binding substance that binds Aβ (x-≥41), but does not bind to Aβ (≥40), and a binding substance that binds tau. In one embodiment, the kit contains four antibodies:

• a) an unlabeled antibody which binds to the junction region of Aβ;
• b) a detectably labeled antibody which binds an epitope containing an amino acid above # 40 in Aβ;
• c) an unlabeled antibody which binds to tau; and
• d) a detectably labeled antibody that binds to tau.

In a further aspect, the present invention provides a system for detecting one of a plurality of Aβ (x-≥41) in a fluid sample. The system includes a first binding substance, typically an antibody specific for an epitope in a binding region of Aβ as described above, and a second binding substance, typically an antibody, specific for an epitope of Aβ containing an amino acid above amino acid no 40 of Aβ at the carboxy terminus, essential for recognition. The first binding substance is an anti-Aβ antibody bound to a solid phase while the other is a reporter antibody to the Aβ carboxy-terminus. The reporter antibody may be self-labeled or may be detectable by another antibody (for example, a rabbit antibody recognizable by labeled or enzyme-conjugated anti-rabbit antibodies). The system may further contain a substrate for an enzyme label. The system is significant in performing enzyme-linked immunosorbent assays (ELISA), which have high specificity and sensitivity for the detection of A (x-≥41) in fluid samples.

In In another aspect, the invention provides methods for screening a connection ready to vote their ability, the crowd of Aβ (x-≥ 41) in the To change CSF. The methods involve measuring a first amount of soluble Aβ (x-≥41) in the CSF of a non-human animal used as a model of Alzheimer's disease; the administration the connection to a non-human animal; measuring a second amount of a soluble Aβ (x-≥41) in the CSF of the non-human animal; and comparing the first amount with the second amount. The difference indicates whether the compound increases Aβ (x-≥41) in the CSF, where In this case they may be significant in the treatment of Alzheimer's can; or the amount reduced, in which case the compound Reinforce Alzheimer's or could accelerate. The non-human animal is preferably a mammal, more preferably a rodent, and most preferably a mouse.

In In another aspect, this invention provides methods for screening a connection ready to determine their ability, the amount from both Aβ (x-≥41) and To change dew in the CSF, involving measuring a first set of one or more soluble Aβ (x-≥ 41) of one non-human animal used as a model of Alzheimer's disease; Measuring a first amount of dew in the CSF of the non-human animal; Administering the compound to a non-human animal; Measuring a second Amount of said one or more soluble Aβ (x-≥ 41) in the non-human CSF animal; Measuring a second amount of tau in the CSF of a non-human animal; and comparing the first amount with the respective second Quantities, the difference indicating whether the compound is the amount soluble Aβ (x- ≥ 41) amplified, diminished or unchanged leaves or the amount of tau in the CSF is increased, decreased or left unchanged. The Information is important, as mentioned above, to identify connections, that can be significant in the treatment of Alzheimer's or which increase Alzheimer's or could accelerate. The non-human animal is preferably a mammal, more preferably a rodent, and most preferably a mouse.

Short description the drawings

1 Figure 12 shows the results of an EILISA assay using antibody 266 (directed at the Aβ junction region) and antibody 277/2 (directed at Aβ amino acid 33-42) to detect Aβ (42) but not Aβ (28). , Aβ (38) or Aβ (40).

2 shows the levels of Aβ (x-≥41) in CSF from control patients (C) and AD patients (AD) in Group A as detected by ELISA.

3 shows the amount of Aβ (x-≥41) in CSF of AD patients (AD) non-Alzheimer neurological controls (NC) and controls (C) in Group B as detected by ELISA.

4 shows the levels of Aβ (x-≥41) in CSF of AD patients (AD), non-Alzheimer neurological controls (ND) and non-dementia controls (NC) as detected by ELISA.

5 shows the levels of tau in CSF of patients with Alzheimer's disease (AD), non-Alzheimer's neurological controls (ND), and non-dementia control patients (NC).

6 shows the levels of Aβ (x-≥41) and tau in CSF of patients with Alzheimer's disease (AD), non-Alzheimer's neurological controls (ND), and non-dementia controls (NC). Data of the 4 and 5 are combined to illustrate the effect of simultaneously considering the two measurements in discriminating the AD group. The lines indicate optimized cupoffs. The high tau / low Aβ (x-≥41) quadrant contains AD patients with only one exception (21 out of 22 patients), with the low tau / high Aβ (x-≥41) quadrant only control individuals contains.

Description of the specific embodiments

The present invention stems, at least in part, from the discovery that the cerebrospinal fluid ("CSF") of individuals suffering from Alzheimer's disease generally contains Aβ (x-≥41) in amounts at the extreme low end of the normal Area lie; which is present in CSF of non-Alzheimer individuals and in particular below 0.5 ng / ml. This discovery is surprising as the The majority of the Aβ deposition in brain tissue of patients suffering from Alzheimer's disease represents Aβ (1-42) and is significantly increased compared to the levels of Aβ (1-42) in non-Alzheimer's individuals.

Based in this discovery, the present invention provides methods for diagnosis and monitoring Alzheimer's Disease. According to a method becomes a Patient sample first. The patient sample is common a liquid sample and preferably a cerebrospinal fluid. Subsequently, will the amount of soluble Aβ (x-≥41) in the Sample of the patient measured. A preferred method for measuring the amount is done by using the sandwich assay as described herein. The measured amount is then compared with a predetermined one Value, such as B. an indicator value in the case of diagnosis or a earlier Patient value in case of monitoring.

Of the Status of the patient is assessed based on the difference between both quantities.

As in greater detail described below, the method of the present invention significant both as a positive and as a negative indicator for AD and other Aβ-related Conditions in tested individuals. The data in the experimental Section show that individuals who are not suffering from Alzheimer's disease suffer from CSF levels of soluble Aβ (x-≥ 41) of 0.2 ng / ml to about 1.0 ng / ml. However, patients with Alzheimer's disease have CSF concentrations of soluble Aβ (x-≥ 41) in general below 0.5 ng / ml. Consequently, the measured amount is above of the indicator value about 0.5 ng / ml a very strong negative indication Alzheimer's disease. That is, individuals with such Mirrors are less likely to be among an Aβ-related Disease, especially with Alzheimer's disease, suffering Need to become.

One Indicator value of 0.7 ng / ml will be the number of false-negative reduce measured values and is also significant as predetermined Amount. In contrast, then a measured amount below of the indicator value 0.5 ng / ml a positive indicator value of Alzheimer's disease and individuals who have these levels are likely to be higher considered to be suffering from Alzheimer's disease. An indicator value of 0.45 ng / ml reduces the number of false-positive values and is also significant as a predetermined value. However, as values below 0.5 ng / ml and 0.45 ng / ml found at the lower end of the normal range in non-Alzheimer's individuals However, a measured value below the indicator level is sufficient not to themselves a diagnosis of Alzheimer's disease available put. Consequently, the methods of the present invention be meaningful as part of a diagnostic procedure that too Consider other known AD symptoms is, such. Those described in the NINCDS-ADRDA criteria (for example, clinical dementia and memory impairment).

The Invention stirs also in part from the discovery that finding Aβ (x-≥ 41) at the bottom End of normal range together with finding higher than normal levels of tau in the individual's CSF are more positive Indicator of Alzheimer's disease compared with the individual findings and a finding of high levels Aβ (x- ≥ 41) and low Levels of dew in the CSF of an individual are a lot more negative Indicator of Alzheimer's disease is. Consequently, the combined use of these two markers seems a significantly complementary one to provide diagnostic information.

The data presented in 6 , show that patients presenting high tau (above about 0.3 ng / ml) and low Aβ (x-≥ 41) (below about 0.5 ng / ml) were 96% likely to have Alzheimer's disease Disease (22/23). 59% of Alzheimer's patients in this study (22/37) fall into this category. In contrast, patients who demonstrated low tau (below about 300 pg / ml) and elevated Aβ (x-≥41) values had a 100% chance of not suffering from Alzheimer's disease (28/28). 6 ). Just over half of non Alzheimer's subjects (28/52, 54%) fell into the category. Taken together, the combined analysis of CSF-tau and -Aβ (x-≥41) of high predictive power for either the presence or absence of Alzheimer's disease was in slightly more than half of the individuals studied in this study. The combined CSF tau and Aβ (x-≥41) measurements were not informative in those patients who fell into the low-Aβ (x-≥41) / low-tau group. Nonetheless, the ability of any assay to aid in the inclusion or exclusion of Alzheimer's disease with high specificity and even moderate sensitivity is of paramount importance.

According to a second method of this invention, the amount of both soluble Aβ (x-≥41) and tau in a patient sample is measured. A significant method for determining the amount of Tau is by ELISA as described in more detail below. The measured amounts of Aβ (x-≥41) and Tau are then compared to predetermined values for the respective size. The status of the patient is evaluated based on the difference between the predetermined values and the measured values.

As discussed below, the indicator value can be calibrated based on the special binding substance that is used and that special Aβ (x-≥ 41) - and Tau protein, that to be detected. The calibration concludes the detection of the binding Substance over Standards of individuals who have an Aβ-related disease, as well as opposite Control standards of individuals who have no such disease. Indicator values are selected based on these results on the number of false-positive or false-negative values, which the user is ready to tolerate. It is expected that indicator values, which use different binding substances which are directed against targets, as described here, roughly equal to the indicator values, as described here, will be. Indicator values below 0.4 ng / ml for Aβ (x-≥ 41) and above 0.4 ng / ml for Tau lower the number of false-positive results; while indicator values above 0.7 ng / ml for Aβ (x-≥41) and below 0.2 ng / ml for Tau the potential for a wrong indication for reduce the absence of Alzheimer's disease.

If the reason for the reduced CSF-Aβ (x-≥41) values in AD actually secondary for the progressing plaque deposition, could be explained why a substantial Number of neurologically ill subjects and few control subjects with low Aβ (x-≥41) levels in CSF showed. There is a hypothesis that the plaque deposition cognitive disorders precedes and a significant portion of this previously non-AD subject might probably AD within the next a few years (DMA Mann et al. (1992) Neurodegeneration 1: 201215 and DL Price et al. (1991) Neurobiol Aging 12: 295-312). Long-term studies are obviously needed for the possibility to address that Aβ (x-≥ 41) levels prognostic character for AD have.

It has also been found that levels of tau in AD-CSF do not correlate with age, MMSE, total Aβ, Aβ ₄₂ or ApoE _E4 . Although the exact reason for the increase in tau in AD remains unclear, it is likely due to the increased levels in AD brain tissue (S. Khatoon et al., (1992) J Neurochem 59: 750-753) combined with the progressive degeneration of AD Neurons in this disease.

Of the Sandwich assay described in the experimental section, used antibody generated against linking region from Aβ and against residues 33-42 from Aβ. In this assay, Alzheimer's patients generally showed levels of Aβ (x-≥ 41) below of 0.5 ng / ml, as by antibody was detected. The indicator value of 0.5 ng / ml is partial a function of the specific peptides recognized by the ones used Antibody, as well as the peptide lot used to perform the calibration. Consequently, can the user set the predetermined amount to a recalibration below Use of reagents and protocols that are used have to, around Aβ (x-≥ 41) in the Prove test. In addition to initial Diagnosis of Aβ-related Condition can the measured concentrations of Aβ are monitored to progress to track the disease and possibly the effectiveness of the treatment to track (if such treatments become available). One would expect that level of Aβ (x- ≥ 41) progresses Illness would fall.

As used herein, the term "amyloid Aβ peptide" or "Aβ" refers to an approximately 4.2 kDa protein which is found in the brains of AD, Down syndrome or HCHWA-D and some normal aged subjects Subunit of amyloid filaments forming the senile (amyloid) plaques and the amyloid deposits in small cerebral and meningeal blood vessels (amyloid angiopathy). Aβ can occur in a filamentous polymeric form (in this form, it shows the congo red and thioflavine S dye-binding characteristics of amyloid described in connection therewith). Aβ may also occur in non-filamentous form ("pre-amyloid" or "amorphous" or "diffuse" deposits) in tissue, in which form there is no detectable birefringent coloration by Congo red. A portion of this protein in the insoluble form obtained from meningeal blood vessels is described in U.S. Patent No. 4,666,829. Aβ is an approximately 39-43 amino acid fragment of a large membrane spanning glycoprotein, referred to as the Aβ amyloid precursor protein (APP), encoded by a gene on the long arm of the human chromosome. Forms of Aβ longer than 43 amino acids are also included here. Aβ is further characterized by its relative mobility in SDS-polyacrylamide gel electrophoresis or in high-performance liquid chromatography (HPLC). A sequence for a 43 amino acid version of Aβ is the following:
1
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
11
Glu Val His His Gln Lys Leu Val Phe Phe
21
Ala Glu Asp Val Gly Ser Asn Lys Gly Ala
31
Ile Ile Gly Leu Met Val Gly Gly Val Val
41
Ile Ala Thr [SEQ ID NO: 1].

As used here, Aβ also refers on related polymorphic forms of Aβ, including those derived from mutations in the Aβ region of the APP normal gene.

Of the Term "Aβ fragment" as used herein refers to fragments and degradation products of Aβ, which in low concentrations of mammalian cells. Special Aβ fragments have a molecular weight of about 3 kD and it is currently believed that they include peptides with, for example, the amino acid residues 3-34, 6-27, 6-34, 6-35, 6-42, 11-34, 11-40, 17-40, 1143 and 12-43 from Aβ.

As As used herein, the term "Aβ (x-≥41)" refers to Aβ whose Aminoterminus with the amino acid No 1 starts from Aβ or which is truncated, with its carboxy terminus on the Amino acid no. 40 goes out. These peptides and fragments include heterogeneous groups. For example, Aβ (6-52), Aβ (11-42) and Aβ (12-43) all found in the CSF. However, this list is not exclusive Thought list. Other peptides that fall under the group exist presumably in the CSF and are using the methods as described here, detectable.

The special peptides measured from within the group of all Aβ (x-≥ 41) depend on the special measuring method used. In the event of the use of binding substances, such as. As antibodies, can the binding substance on one or more within the group be directed by peptides. For example, an antibody is generated against amino acid 33-42 from Aβ, which does not crossreact with Aβ (1-40) bind to Aβ (x-42). He may also respond to Aβ (x-41) and Bind Aβ (x-43). According to one embodiment the invention concludes the method comprises determining the amount of Aβ (x-≥41) with at least amino acids 13-41 of Aβ. This species can be measured using a sandwich assay, the antibody which uses the linking region (Amino acids 13-26) recognize and antibodies produced by immunization with a hapten with amino acids 33-42 as described in the example.

The term "Aβ linking region" as used herein refers to a region of Aβ whose midpoint is at the site of amino acid residues 16 and 17 (Lys ¹⁶ and Leu ¹⁷ ), which is a target for the proteolytic processing of APP. Such processing results in a variety of APP fragments which, for example, terminate at amino acid 16 of Aβ, and which consequently are potentially immunologically cross-reactive with antibodies to the intact Aβ molecule to be identified in the method of the present invention. Antibody generated against a synthetic peptide, including amino acid residues 19-29, could be found to show the necessary specificity.

Of the Term "amyloid β-precursor protein" (APP), as here is defined as a polypeptide that is linked by a gene of the the same name is encoded and in people on the long arm from chromosome 21 and which Aβ is located within its carboxyl third includes. APP is a glycosylated spanning a single membrane Protein expressed in a large variety of cells in many mammalian tissues. Examples of specific isotypes of APP currently known to be they exist in humans are described by the 695-amino acid polypeptide of Kang et al. (1987) Nature 325: 733-736, referred to herein as the "normal" APP; the 751-amino acid polypeptide, described by Ponte et al. (1988) Nature 331: 525-527 (1988) and Tanzi et al. (1988) Nature 331: 528-530; and the 770 amino acid polypeptide, described by Kitaguchi et al. (1988) Nature 331: 530-532.

Examples specific variants of APP include point mutations that Both in position and phenotype can distinguish (for a Review concerning known variant mutations see Hardy (1992) Nature Genet. 1: 233234).

The Name "Aβ-related Condition ", as here is defined as including Alzheimer's disease (which family Including Alzheimer's disease), Down syndrome, HCHWA-D and advanced brain aging.

As Used here, "dew" refers to the family of microtubule-associated proteins. The paired helical Filaments of neurofibrillary tangles in the brain of a patient with Alzheimer's disease from the dew protein. (See, for example, M. Goedert et al. (1989) Neuron 3: 519-526 and M. Goedert (1993) TINS 16: 460-465) Goedert et al. (1989) presents also a DNA and an amino acid sequence with dew.

Of the Term "body fluid" as used herein concerns those liquids a mammal host, which will be expected to contain detectable levels of Aβ, Aβ fragments or containing tau protein, specifically including cerebrospinal fluid (CSF), blood, urine and peritoneal fluid. The term "blood" refers both on blood as a whole as well as on blood plasma and serum.

The Methods and systems of this invention include the ability to detect species of Aβ, which over amino acid No. 40 at the carboxy-terminal end, and consequently, the Ability, from shorter ones Species, such as B. Aβ (40) to distinguish. While the detection of Aβ (x-≥ 41) any methods known in the art for detection can be realized by peptides, is the application of immunological Detection techniques that detect binding substances such as antibodies, antibody fragments, recombinant antibodies and the like, include prefers. Particularly suitable detection techniques include ELISA, Western blotting, radioimmunoassay and the like. Suitable immunological Procedures that include a single antibody will become also considered, for example, a radioimmunoassay using a specific antibody against ≥ 41 shapes from Aβ; or Single antibody ELISA methods.

consequently This invention also provides antibodies that are specific for Aβ (x-≥ 41), which do not cross react with Aβ (≥ 40).

These antibody can be prepared by immunizing animals with synthetic Peptides, which amino acids contained above No. 40 of Aβ. For example, the synthetic peptide may include amino acids 33-42. One specific example of the production of such an antibody is experimental Section available posed.

According to one embodiment The invention involves the detection and measurement of Aβ (x-≥41) peptides the use of two antibodies, a specific for an epitope containing amino acids via No. 40 in Aβ addition and another antibody, which is capable of Aβ and Aβ fragments different from other APP fragments that may be to find in a sample. In particular, it has been found that antibodies that monospecific for the linking region are from Aβ, are able to Aβ from others To distinguish APP fragments. The linking region of Aβ has its Middle at the amino acid residues 16 and 17, typically spanning amino acid residues 13-26, and such linking-specific antibody can be prepared using synthetic peptides with the Sequence as an immunogen.

A preferred immunoassay technique is a two-site or "sandwich" assay that includes a linking specific antibody as the capture antibody (bound to a solid phase) and a second antibody, the an epitope which contains the amino acids via No. 40 in Aβ. Specific methods for making and using such antibodies antibody in an exemplary ELISA will be in an experimental section shown below and in the related US patent application 07 / 965,972, supra.

Antibody specific for Aβ can be made against a suitable antigen or hapten comprising the desired target epitope, such as B. the linking region, consisting of amino acid residues 13-29 and the carboxy terminus consisting of amino acid residues 33-42. conveniently, can the synthetic peptides by conventional solid-phase techniques prepared, coupled to a suitable immunogen and used be to antisera and monoclonal antibodies by conventional techniques manufacture. Suitable peptide haptens are usually at least five adjacent Include residues of Aβ and can more than six residues.

synthetic Polypeptidhaptene can prepared by the well-known Merrifield solid-phase synthesis technique, in which amino acids be added sequentially to a growing chain (Merrifield (1963) J. Am. Chem. Soc. 85: 2149-2156). The amino acid sequence can be on the sequence of Aβ, like shown above.

Once a sufficient amount of polypeptide hapten has been obtained, it can be conjugated to a suitable immunogenic carrier, such as e.g. Serum albumin, keyhole limpet hemocyanin or other suitable protein carriers as generally described in Hudson and Hay, Practical Immunology, Blackwell Scientific Publications, Oxford, Chapter 1.3, 1980. An exemplary immunogenic carrier used in the Examples as provided below , is the α-CD3 _E antibody (Boehringer-Mannheim, clone No. 145-2C11).

As soon as a sufficient amount of the immunogen has been obtained, antibodies specific for desired epitope produced by in vitro or in vivo techniques. in vitro techniques shut down the exposure of the lymphocytes to immunogens while in In vivo techniques include injection of the immunogens into suitable vertebrate hosts desire. Suitable vertebrate hosts are not human, including mice Rats, rabbits, sheep, goats and the like. Become immunogens injected into the animals according to a predetermined pattern and the animals are periodically bled, the sequence of blood fractions having improved titers and specificity. The injections can intramuscularly, carried out intraperitoneally, subcutaneously or the like and an adjuvant, such as B. the incomplete Freund's adjuvant can be used.

If required can monoclonal antibodies to be obtained by dissecting of immortalized cell lines that are capable of carrying antibodies desired specificity to create. Such immortalized cell lines can be generated be in different ways. Conveniently, a small Vertebrate, such. As a mouse, hyperimmunized with the desired Immunogen by the method just described. The vertebrate is then killed, usually a few days after the last immunization. The cells of the spleen are removed and the cells of the spleen are immortalized. The Way of immortalization is not critical. Currently is the most common Technique fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1975) Nature 256: 495-497. Other techniques shut down the EBV transformation, transformation with naked DNA, for example through oncogenes, retroviruses, etc., or any other method, for the stable maintenance of the cell line and for the production of monoclonal antibody contribute. Specific techniques for producing monoclonal antibodies will be described in Antibodies: A Laboratory Manual, Harlow and Lane, Ed., Cold Spring Harbor Laboratory, 1988.

In addition to the monoclonal antibodies and the polyclonal antibodies (antisera), the detection techniques of the present invention will also be capable of employing antibody fragments, e.g. F (ab), Fv, V _L , V _H and other fragments. However, with the use of polyclonal antibodies, it may be necessary to adsorb the antisera to target epitopes to create a monospecific antibody population. It will also be possible to use recombinantly produced antibodies (immunoglobulins) as well as variants thereof, as well described in the patent and scientific literature. See, for example, EPO 8430268.0; EPO 85102665.8; EPO 85305604.2; PCT / GB 85/00392; EPO 85115311.4; PCT / US86 / 002269; and Japanese Application No. 85239543. It would also be possible to produce other recombinant proteins that would mimic the binding specificity of antibodies prepared as just described.

The Detection of dew can also be realized by any Methods known in the art for detecting peptides are known. The use of immunological detection techniques, however, which uses binding substances is preferred. significant Detection techniques close all the above one. ELISA assays involving a trapping antibody and a labeled detection antibody, both against Tau especially significant.

Antibodies against Dew can are produced by vaccinating the animals with dew, purified AD brains or from recombinant sources. Recominant rope can can be generated by expression in insect cells from a baculovirus vector, containing the pVL941-Tau-4 repeat isoform as described by J. Knops et al. (1991) J. Cell Biol 1991: 114: 725-733. Purified dew is also available by Immogenetics (Zwijndrecht, Belgium). Antibodies to Tau are also available from Sigma (St. Louis, MO). Next sources can be identified in the Lindscott dictionary.

Tau can be generated from AD brain by the method of Mercken et al. (1992) J Neurochem 58: 548. Typically, 50 g of fresh brain are cut into small pieces with scissors and 1: 1 (w / v) in buffer A (20 mM 2- [N-morpholino] ethanesulfonic acid, 80 mM NaCl , 2mM EDTA, 0.1mM EGTA, 1mM MgCl ₂ and 1mM β-mercaptoethanol, pH 6.75) were homogenized with a Potter homogenizer equipped with a Teflon flask. The homogenate is centrifuged for 1 hour at 150 000 g at 4 ° C and the over is heated for 5 minutes in boiling water and cooled again on ice for 10 minutes. The slurry is centrifuged for 2 hours at 150,000g at 4 ° C and the supernatant is then collected. The heat-stable cytosolic extract is acidified with 2.5% perchloric acid and centrifuged for 1 hour at 150,000 g at 4 ° C, after which the supernatant is neutralized with 3 M Tris. The supernatant is then dialyzed and concentrated in water in a Centiprep concentrator (Amicon, Lausanne, Switzerland). The final product can be evaluated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli method). This method is important for immunizing animals to produce anti-tau antibodies.

dew can also be immuno-purified from this preparation. ten Milligrams of anti-tau monoclonal antibody are activated on 1 g of cyanogen bromide Sepharose (Pharmacia) by the method described by the manufacturer, coupled. Fifty Milliliters of the heat stable cytosolic extract as described above are diluted 1: 2 in 0.1 M phosphate buffer (pH 8.5) and on the pillar applied. The pillar is washed with 0.1 M phosphate and tau is treated with 0.1 M citric acid (pH 2.5) and immediately neutralized with 1 M NaOH. The fractions can evaluated by SDS-PAGE on 10% gels and by immunoblotting with anti-tau antibodies.

These The invention also provides kits for performing the assays which helpful in the diagnosis of Alzheimer's disease. The kits shut down Means for detecting Aβ (x-≥ 41) and Means for detecting dew. These agents may include any agents that are known or described above, for example, binding Substances. Significant binding substances include molecules, which have the binding portion of an antibody, such as. B. a whole antibody or antibody fragments. The binding substances can monoclonal antibodies be. In one embodiment includes the kit incorporates a binding substance that binds Aβ (x-≥41), but does not Aβ (≥40) binds, and a binding substance that binds to tau.

In an embodiment includes the kit antibodies or the like, for performing from sandwich ELISAs to any connection, such as described above. In one embodiment, the Means for detecting Aβ (x-≥ 41) one include binding substance, which binds to an epitope containing amino acids above # 40 in Contains Aβ, as well as a binding substance, which Aβ or binds a fragment of Aβ, however, does not bind other fragments of APP. The means to detect of dew can also include a sandwich ELISA. For example, the Kit a) an unlabeled binding substance attached to a linking region of Aβ binds, lock in; b) a detectable labeled binding substance attached to an epitope binds the amino acids above No. 40 in Aβ; c) one unlabeled binding substance that binds to tau; and d) one detectable labeled binding substance that binds to tau.

The detectable labels be any known and used in the art, inclusively for example, biotinylation, radioactive labels, enzymes, fluorescent Label and the like.

animal models are currently being used to study Alzheimer's disease. (see for example international patent application WO 93/14200, US Patent Application 08 / 143,697, filed October 27, 1993 and US Patent 5,387,742.) These models are important for screening compounds on theirs Ability, to influence the course of Alzheimer's disease, both in terms of improvements as well as deterioration of the condition. Since AD is characterized by the decrease in the amount of Aβ (x-≥ 41) in the CSF, one expects to find effective treatments for Alzheimer's disease in one Increase in the amount of Aβ (x-≥41) in the CSF results, however, agents that alter the course of the disease will result in the decrease in the amount of Aβ (x-≥41) in the CSF.

Accordingly, the present invention provides methods for screening compounds which increase or decrease the amount of Aβ (x-≥41) in a fluid sample, particularly in CSF, and which are therefore candidates for use in the treatment of the disease or which accelerate the disease and should therefore be avoided by humans. The methods include measuring a first amount of said one or more soluble Aβ (x-≥41) in a non-human animal sample used as a model for Alzheimer's disease; administering the compound to the animal; measuring a second amount of one or more soluble Aβ (x-≥41) in a sample of the animal; and comparing the first amount to the second amount, the differences indicating whether the compound increases, decreases or leaves unchanged the amount of soluble Aβ (x-≥41) in the sample. The degree of dosage administered to the animal and the amount of time that passes before the second amount will be measured will naturally depend on the model system.

These Invention also provides methods for screening compounds which increase the amount of Aβ (x-≥41) and the amount of dew in a fluid sample lower, especially in CSF, and thus candidates for Application in the treatment of the disease are; or which the mirrors of Aβ (x-≥ 41) and which increase the levels of dew and thus speed up the disease and should be avoided by humans. Involve the procedures measuring a first quantity of said one or more soluble ones Aβ (x-≥41) and Tau in a fluid sample a non-human animal used as a model for Alzheimer's disease; Administering the compound to the animal; Measuring a second quantity of one or more soluble Aβ (x-≥41) and Tau in a fluid sample of the animal; and comparing the first quantities with the second quantities, the difference indicating whether the compound is the amount of soluble Aβ (x-≥41) and Tau in the liquid Sample increased, diminished or unchanged leaves. The degree of dosage administered to an animal and the amount of Time that elapses before the second reading will be measured, become natural depend on the model system.

A significant non-human animal model harbors a copy of an expressing bar gene transgene encoding the Swedish mutation of APP (asparagine ⁵⁹⁵ -leucine ⁵⁹⁶ ). The sequence is generally expressed in cells that normally express the naturally-occurring endogenous APP gene (if available). Mammalian models, more specifically rodent models, and especially mouse and hamster models, are suitable for this application. Such transgenes typically include a Swedish mutation APP expression cassette in which a linked promoter, and preferably an enhancer, express expression of structural sequences; encoding a heterologous APP polypeptide comprising the Swedish mutation.

The transgenic animals harboring the transgene, which is a Swedish Mutation APP polypeptide encoding are usually by inserting the Transgene or targeting construct into a fertilized egg or embryonic stem (ES) cell, typically by microinjection, Electroporation, lipofection or biolistic produced. The transgenic Animals express the Swedish mutation APP gene of the transgene (or the homologous recombinant target construct), typically in brain tissue. Preferably, one or both are endogenous APP alleles inactivated and unable to express the wild-type APP.

The following examples are offered for illustration, not for Limit.

Examples

I. Aβ (x-≥41) is in Alzheimer's patients reduced

Materials and methods

1. Antibody production

a. Monoclonal antibodies against the Aβ junction region

Monoclonal antibodies to the junction region of Aβ were prepared using a synthetic peptide spanning amino acid residues 13-31, except that AI, amino acids 30 and 31, were substituted with GC. This peptide was named Aβ _13-28 . Aβ _13-28 was linked to an immunogen (α-CD3 _E antibody, clone # 145-2C11, Boehringer-Mannheim) using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) according to the manufacturer's instructions (Pierce ).

A / J mice were initially immunized intraperitoneally (IP) with the Aβ conjugate mixed with complete Freund's adjuvant. Fourteen days later, immunization in the mice was boosted IP with the Aβ conjugate mixed with phosphate buffered saline (PBS) at 14-day intervals. After six complete boosts, the immunization in the mice was ultimately boosted intravenously with the Aβ conjugate mixed with the Freund's incomplete adjuvant, and the mice were fused 3 days later. Fusion of spleen cells with P3.653 myeloma cells was performed as described in Oi and Herzenberg, Selective Methods in Cellular Immunology, Mishell and Shigii, Eds., WH Freeman and Company, San Francisco, Chapter 17 (1980). Some titers and initial screens were performed by the RIA method as described below. Some clones were extended to a 24-well plate and subjected to further analysis as described below. Clones of interest were found in mouse ascites provides.

The RIA method used to screen serum blood fractions and fusion hybridoma supernatants was based on a method developed by Wang et al. (1977) J. Immunol. Methods 18: 157-164. Briefly, the supernatant (or serum) was incubated overnight at room temperature on a rotator with ¹²⁵ I labeled Aβ _1-28 and Sepharose 4B beads to which sheep anti-mouse IgG had been coupled via cyanobromide. The beads of each well were harvested onto glass fiber filter discs with a cell harvester and washed several times with PBS. The filter discs were then transferred to gamma tubes and the bound radioactivity was counted with a gamma counter.

All hybridomas were tested for binding to Aβ _1-28 using the procedure described above in the initial screen, and then tested again 3 days later. The Aβ _1-28 positive clones were further characterized for reactivity with ¹²⁵ I-labeled Aβ _1-16 using the RIA method as described above. No clone was identified binding to Aβ _1-16 . In a peptide capture ELISA, all clones reacted with Aβ _13-28 , whereas no clone reacted with Aβ _17-28 . Thus, it was determined that all clones had an epitope within the linking region spanning amino acids 16 and 17.

Based on the results of the above assays, additional clones were extended to the 24-well plates. These clones were further characterized by saturation analysis. Supernatants at the 50% -Titerpunkt (as determined as described above by the RIA method, was) was added to the wells, which Sepharose ^® -Schaf-anti-mouse IgG beads, a constant amount of ¹²⁵ I-of labeled Aβ _1-28 and varying amounts of unlabeled Aβ _13-28 or Aβ _17-28 . The concentration of cold peptide for 50% inhibition was determined for each antibody. For Aβ _17-28 , no inhibition was found at 100 ng / well for any clone. The 50% inhibition point for Aβ _13-28 was on the order of 10-80 ng / well. The clones were also characterized based on reactivity in Western blots. Based on the titer point, the sensitivity (as determined by the 50% inhibition point) and the reactivity on Western blot, some clones were produced in ascites. Hybridoma antibodies, termed 266, were selected as capture antibodies in the assays described below.

b. Polyclonal antibodies against the C-terminal epitope containing amino acids 33-42 of Aβ

Polyclonal antibodies were raised against Aβ (33-42) as follows. Peptide 277-2 (C-aminoheptanoides-GLMVGGVVIA [SEQ ID NO: 2]) was conjugated to cationized BSA (Pierce activated "supercarrier") in a ratio of 5 mg of 277-2 peptide to 10 mg of cationized BSA as follows. A vial of Pierce supercarrier (10 mg) was resuspended in 1 ml of deionized water. 5 mg of the 277-2 peptide was dissolved in 5 ml of 10 mM PO _4, pH 8.0, dissolved. The 277-2 peptide was added to the supercarrier and incubated overnight at room temperature. This was then concentrated and the EDTA removed.

The Immunogen (500 mg of peptide equivalent) was subcutaneously in complete Freund adjuvant injected. Rabbits received a boost of 0.2 to 0.5 mg after three weeks and 0.2 to 0.5 mg after two to four weekly intervals thereafter. The reinforcements were subcutaneously in incomplete Freund's adjuvant administered. twenty-five ml of serum was collected one week after each augmentation. blood fractions were screened as follows. Blood fractions of week 7 of rabbits were through serial dilution titered. The ELISA plates were coated with Aβ 1-42 overnight and then blocked with 3% gelatin. Serial dilutions the rabbit blood fractions of 1 / 100-1 / 200,000 were on the plates for 2 hours incubated at room temperature. The plates were then washed and the anti-rabbit HRP was added to each well. This incubated for 1 hour. The plate was washed and the TMB substrate became used. The ELISA titer of the rabbits was 1/20 000-1 / 200,000.

The ELISA-positive rabbit blood fractions were then titered in a capture RIA to compare their ability to bind ¹²⁵ I Aβ (1-42) to ¹²⁵ I Aβ (1-40). Dilutions of rabbit anti-serum from 1 / 25-1 / 675 were incubated with approximately the same number of cpm from both tracers. Protein A-Sepharose was used to precipitate the immune complexes and these were then counted on a Microbeta scintillation counter. 277-2 Rabbit D shows the highest titer against Aβ (1-42) tracer and no cross reaction with Aβ (1-40) tracer. The highest titer blood fractions were then subjected to affinity purification of antibodies.

To purify the anit 277-2 antibody affinity, a 277-2 affinity matrix was prepared as follows: Three mls of sulfo-linked gel (Pierce) were mixed with six volumes of 50 mM Tris, 5 mM EDTA, pH 8.5, washed. Three mg of 277-2 peptide dissolved in 0.3 ml of DMSO was made up to 3 ml with 50 mM Tris, 5 mM EDTA at pH 8.5 and added to the gel. After mild mixing for 15 minutes, the column resin was washed with six volumes of 50 mM Tris, 5 mM EDTA, 0.5 M NaCl at pH 8. The columnar resin was then washed with 16 volumes of PBS / 0.05% NaN ₃ .

To affinity-purify the antibodies, 20 ml of high titre serum was diluted to 40 ml with PBS and an equal volume of saturated (NH ₄ ) ₂ SO ₄ was slowly added with stirring at 4 ° C. The mixture was allowed to stir for a further 30 minutes and then spun for 15 minutes at 10,000 rpm in a Beckman JA17 rotor. The precipitates were resuspended in PBS, brought to a volume of 40 ml with PBS and the (NH ₄ ) ₂ SO ₄ precipitate reprecipitated as above. The precipitates were resuspended in 20 ml total of PBS and dialyzed overnight against PBS at 4 ° C.

The 277-2 column was washed with 10 ml of PBS. Subsequently, the dialysate was passed over the column. The pillar was then washed with 50 ml of PBS. 0.1 M glycine, 0.5 M NaCl, pH 2.5 was added to 1 ml at a time and the fractions were collected. The first four fractions containing the majority of the eluted protein were pooled and 0.4 ml of 1 M Tris neutralized at pH 8.0. The pool was concentrated by membrane filtration to just under 2 ml. The original column flow became one second chromatographic step (after first the Neutralized column had been reequilibrated in PBS had been). The second affinity purified Material became more similar Neutralized and concentrated, combined with the first material and then dialyzed against PBS at 4 ° C. The protein content was determined (Pierce BCA method) and this antibody were used in ELISA experiments.

2. ELISA assay

a. Binding catching antibodies on microtiter wells

Monoclonal antibody 266 was diluted to a concentration of 10 μg / ml in a buffer containing 0.23 g / l NaH ₂ PO ₄ H ₂ O, 26.2 g / l Na ₂ HPO ₄ ; 7H ₂ O, 1 g / l NaN ₃ , pH 8.5. One hundred μl / well of this solution was then distributed in a 96-well white Dynatech Microlite 2, 96-well flat-bottomed plate. The plates were sealed and incubated overnight at room temperature. After coating, the remaining solution was aspirated and the non-specific binding sites were added to 200 μl per well (NaH ₂ PO ₄ H ₂ O) 0.2 g / l, Na ₂ HPO ₄ .7H ₂ O 0.8 g / l , human serum albumin (NSA) and lyophilized, 2.5 g / L, pH 7.4. These plates were blocked by incubating for 1 hour at room temperature in the blocking solution.

b. assay protocol

The calibrators were prepared from a stock solution of Aβ _1-42 , 1 μg / ml in DMSO. In the sample dilution ((NaH ₂ PO ₄ H ₂ O) 0.2 g / l, Na ₂ HPO ₄ .7H ₂ O 2.16 g / l, NaN ₃ 0.5 g / l, bovine serum albumin (BSA) (globulin-free 6 g / l, Triton x-405 0.5 ml / l NaCl 8.5 g / l, pH 7.4) was the highest calibrator, 1000 pg / ml (10 μl Aβ _1-42 strain (1 μg / ml DMSO) in 10 ml casein sample dilution). Subsequent dilutions were made in the sample dilution to obtain 500, 250, 125, 62.5 and 31.25 pg / ml concentrations of Aβ _1-42 .

CSF samples were prepared as follows. The CSF samples (100-500 μl) were for 3 minutes cooked. The boiled samples were taken at 4 ° C for 10-14 hours before performing the Placed assays. CSF samples were assayed neat. dilutions were only made if the originally calculated value was above of the highest Calibrator was (1000 pg / ml).

One hundred μl per well Calibrators or samples were applied to the microtiter plate. The plates were sealed and left for 1 hour at room temperature incubated. The plates were then washed three times with washing solution (NaCl 80 g / l, KCl 3.85 g / l, Tris-HCl 31.75 g / l, Tween-20 0.5 ml / l, pH 7.5).

Anti-Aβ (33-42) (antibody 277-2) was diluted to 1 μg / ml in sample dilution and 100 μl was added per well. The plate was covered and incubated for 1 hour at room temperature. The plate was washed three times with washing solution. The alkaline phosphatase affinity purified F (ab ') ₂ fragment of donkey anti-rabbit IgG (H + L) (Jackson) was diluted 1: 1000 in sample dilution. One hundred μl / well was added. The plate was covered and incubated for 1 hour at room temperature. The plate was washed three times with washing solution and then 100 μl / well of a che added miluminescent substrate. The chemiluminescent substrate was prepared by diluting the chemiluminescent reagent, AMPPD (Tropix) and an enhancer, Emerald green (Tropix), 1: 1000 and 1: 100 respectively in 1 M diethanolamine buffer, pH 10, containing 1 mM MgCl ₂ and 0.2%. NaN ₃ produced. The plates were sealed and incubated for 10 to 15 minutes at room temperature. The solution was not aspirated. This time may need optimization as far as other antibody lots are concerned.

chemiluminescence was read out and as in the form of relative chemiluminescent units (chemiluminescence units, CLU) after 15 minutes using of a Dynatech ML 1000.

Results

1. Aβ (x-≥41) assay specificity

Aβ (x-≥41) ELISA does not cross react with Aβ (1-28), (1-38) and (1-40) ( 1 ).

2. Aβ (x-≥41) assay sensitivity

The lower sensitivity limit for this assay is 31 pg / ml or 3.1 pg / well (1.7 fmol / well ( 1 ).

3. Aβ (x-≥41) level in CSF

Aβ (x-≥41) was verified in CSF using the Aβ (x-≥41) ELISA. From time to time, two different groups of CSF samples called Group A and Group B were obtained from different sources. Sometimes, two hundred μl of the CSF samples were boiled for 3 minutes before the assay (it was found that boiling increased the Aβ (x-≥41) immunoreactivity in some cases). The results of this assay can be found in 2 and 3 be seen. Table 1 summarizes these results.

4. Aβ (x-≥41) in CSF of rodents and dogs

Aβ (x-≥41) immunoreactivity also detected in CSF of guinea pigs and dogs. (Table II).

These Sandwich ELISA demonstrates the presence of Aβ (x-≥41) in CSF. Aβ (x-≥ 41) is only a minor component of the total amount of Aβ in CSF. The levels of Aβ (x-≥41) in CSF are significantly lower in AD than in normal and neurological Controls. Assuming a 50% sensitivity limit, the specificity is 93.8 for group A and 97.4% for Group B. These two independent Groups show a noticeable similarity, which demonstrates that the measurement of Aβ (x-≥41) in CSF has diagnostic significance.

II. Combined measurements of Aβ (x-≥41) and Tau are highly sensitive for Alzheimer's disease

Materials and methods

1. subjects

• i. AD (n = 37): Die Patienten erfüllten die NINCDS-ADRDA-Guidelines für wahrscheinliches AD; diejenigen, welche Kriterien für mögliches AD erfüllten, wurden ausgeschlossen (The Lund and Manchester Groups (1994) J Neurol. Neurosurg Psychiatr 57: 416–418). Alle Patienten wohnten in Gemeinschaftsunterkünften und zeigten milde bis moderate Demenz.
• ii. Neurologisch erkrankte Kontrollen (ND; n = 32): Patienten mit Nicht-AD-Demenz oder degenerativen Erkrankungen, welche das zentrale Nervensystem betreffen. Für neurologische Kontrollen wurde eine Zusammenfassung von klinischen Aufzeichnungen durch einen zweiten Neurologen (DG) überprüft, um die Diagnose zu bestätigen, und sicherzustellen, dass gleichzeitig existierendes AD unwahrscheinlich war. Patienten mit Stirnhirndemenz wurden gemäß den Kriterien, wie dargestellt von der Lund- und der Manchestergruppe diagnostiziert (Kawasaki E. S., in: PCR Protocols: A guide to methods and applications. Academic Press, Inc., New York 1990, S. 146–152).
• iii. Nicht-Demenz-Kontrollen (NC; n = 20): Die Subjekte waren 50 Jahre oder älter und wiesen keine signifikanten kognitiven Beschwerden auf, zeigten keine funktionellen Ausfälle, hatten normale Befunde bei der neurologischen Überprüfung und erreichten 28–30 auf dem MMSE. Eine Subgruppe dieser Kontrollen zeigte Symptome der Depression, die nicht in signifikantem kognitiven oder funktionellen Schädigungen resultierte und wurde als Nicht-AD oder irgendeine andere organische neurologische Erkrankung aufweisend eingestuft. Rückenmarkspunkturen wurden am Morgen nach Übernacht-Abstinenz genommen. Alle CSF-Proben wurden in Probenröhrchen, verteilt an alle Stellen, eingesammelt. Die ersten 2–3 ml der CSF wurden hinsichtlich Protein, Glucose und Zellen am örtlichen Laboratorium des medizinischen Zentrums analysiert und 4,5 ml wurden von ursprünglich gesammelten Röhrchen entfernt und zu 8 ml Sarstedt-Röhrchen, enthaltend 500 μl Puffer gegeben (enthaltend Additive, wie z. B. der letztendlichen CSF-Lösungs-Zusammensetzung mit folgenden Bestandteilen: 20 mM Natriumphosphat, 20 mM Triethanolamin, 0,05% Triton X-100, 100 mM NaCl, 0,05% NaN₃, 1 mM Diethylentriaminpentaessigsäure, 1 mM EGTA, pH 7,4) und wurden bei –20°C bis zur Analyse eingefroren. Die Assayoperatoren kannten die Diagnosen des jeweiligen Subjektes nicht.

All subjects included in this study were subjected to a detailed clinical and neurological examination at medical centers of universities by experts in the field of neurology for the diagnosis of dementia. Declarations of consent were received from the subjects or, if appropriate, the guardian. The assessment included medical history, physical and neurological examinations, laboratory blood tests to rule out metabolic causes of dementia, neuroimaging imaging (head CT or MR within the past 3 years for patients with dementia and neurological controls) and detailed psychometric tests (these varied between institutions). In addition, all subjects received the following assessment tools: Mini-Mental State Examination (MMSE) (American Psychiatric Association, Committee on Nomenclature and Statistics: Diagnostic and Statistical Manual of Mental Disorders: Revised Third Edition, Washington DC Psych. Associ., (1987)), the Hamilton Depression Inventory (VC Hachiniski et al., (1975) Ann. Neurol., 32: 632-637) and the Hachinski ischemic index (McKann, G., et al., (1984) Neurology 34: 939-944). Patients with more than one dementia diagnosis, recent stroke, head trauma, or significant peripheral nervous system disorders have been excluded. The following diagnostic criteria were used:

• i. AD (n = 37): Patients met the NINCDS ADRDA guidelines for probable AD; those that met criteria for possible AD were excluded (The Lund and Manchester Groups (1994) J Neurol, Neurosurg Psychiatr 57: 416-418). All patients lived in shared accommodation and showed mild to moderate dementia.
• ii. Neurologically impaired controls (ND; n = 32): Patients with non-AD dementia or degenerative diseases affecting the central nervous system. For neurological controls, a summary of clinical records was reviewed by a second neurologist (DG) to confirm the diagnosis and to ensure that concomitant AD was unlikely. Patients with cerebral cerebral dementia were diagnosed according to the criteria as outlined by the Lund and Manchester groups (Kawasaki ES, in: PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc., New York 1990, pp. 146-152). ,
• iii. Non-Dementia Controls (NC; n = 20): The subjects were 50 years or older and had no significant cognitive discomfort, showed no functional failure, had normal findings at the neurological examination and reached 28-30 on the MMSE. A subgroup of these controls showed symptoms of depression that did not result in significant cognitive or functional damage and was classified as having non-AD or any other organic neurological disorder. Spinal cord punctures were taken the morning after overnight abstinence. All CSF samples were collected in sample tubes distributed to all sites. The first 2-3 ml of CSF were analyzed for protein, glucose and cells at the local laboratory of the medical center and 4.5 ml were removed from originally collected tubes and added to 8 ml Sarstedt tubes containing 500 μl of buffer (containing additives such as the final CSF Solution composition comprising: 20 mM sodium phosphate, 20 mM triethanolamine, 0.05% Triton X-100, 100 mM NaCl, 0.05% NaN ₃ , 1 mM diethylenetriamine pentaacetic acid, 1 mM EGTA, pH 7.4) and were frozen at -20 ° C until analysis. The assay operators did not know the diagnoses of the subject.

2. ApoE genotyping

The ApoE genotyping was performed with available blood samples, collected in ED-TA Vacutainer tubes were. The samples were prepared by the method of Kawasaki (Kawasaki ES, in: PCR Protocols: A guide to methods and applications, Academic Press, Inc., New York 1990 pp. 146-152) and the PCR analysis was carried out as described by Wenham (P.R. Wenham et al. (1991) Lancet 337: 1158-1159).

3. Total Aβ ELISA

The Total amount of Aβ was in a sequential double monoclonal antibody sandwich ELISA measured as described in Seubert et al. (1992) Nature 359: 325-327. Briefly, Aβ was in CSF by the monoclonal antibodies 266 captured (specific to Aβ-peptide residues 13-28), previously coated in microtiter plate wells. The proof set a second Aβ-specific, biotinylated monoclonal antibody 6C6 (which recognized Aβ residues 1-16), followed by reactions with an alkaline phosphatase-avidin conjugate. After incubation with the fluorogenic substrate 4-methyl-Umbellipherylphosphat (MUP) was the fluorescent product using a Millipore Cytofluor 2350 fluorometer.

4. Aβ (x-≥41) ELISA

Aβ (x-≥41) was measured in a similarly formatted assay using 266 as the capture antibody. Polyclonal reporter antibody 277-2 was raised against a synthetic peptide containing Aβ residues 33-42 (GLMVGGVVIA) [SEQ ID NO: 2] with cysteine-aminoheptane acid at its amino terminus. It was conjugated to cationized BSA (Pierce) by the conjugated cysteine. Antibody 277-2 was affinity purified using the synthetic peptide conjugated to a sulfo-linked acid (Pierce) and reacted strongly with ¹²⁵ I-Aβ _1-42 as detected by precipitation of the tracer. It showed no detectable cross-reactivity with Aβ _1-40 neither in immunoprecipitation nor in the ELISA formats, suggesting that there is at least a 1000-fold reduced sensitivity to the Aβ _1-40 peptide. Synthetic Aβ _1-42 was used as standard. Detection of the 277-2 reporter antibody was achieved using a donkey anti-rabbit IgG alkaline phosphatase conjugate and the AMPPD chemiluminescent substrate with the emerald enhancer (Tropix) (Vigo-Pelfrey, C., et al J Neurochem 61: 1965-1968).

Around the inter-assay variability as a factor in Aβ (x-≥41) analysis ruled out All samples were taken twice on the same day with the same batch went to standards. The intraassay variability was less than 10%. In front In the measurement, aliquots of CSF samples were heated to 100 ° C for 3 minutes heated and then at 4 ° C overnight stored before the assay. It turned out that the heating step generally increased the immunoreactivity in CSF samples and Although independent from the diagnosis and therefore it was included. It should It can be noted that different batches of synthetic Aβ (x-≥ 41) are light produce different default values, although by amino acid analysis were normalized. The listed Values are based on a single standard that applies to the entire Investigation was used. Investigations on the addition of synthetic Aβ (x-≥41) to CSF included, demonstrated that the measured recognition 80 ± 50% amounted to.

5. proof of tau by ELISA

a. Purified dew

Tau, purified from human AD brain tissue and from recombinant sources were used to characterize the assay and the antibodies. Recombinant human tau was generated using the baculovirus vector described previously containing the pVL941-tau-4 Re peat isoform (J Knops et al. (1991) J Cell Biol 1991: 114: 725-733). High levels of tau were expressed and purified from both SF9 and high five insect cells. Maximum expressing cell cultures were harvested, washed once in PBS and chilled on ice. The cells were then disrupted by ultrasound in 0.1M MES pH 6.5, 1mM EGTA, 18μM EDTA, 0.5mM MgCl ₂ , 5μg / ml Leupeptin, 1mM PMSF. Cell deposition was removed by low speed centrifugation and the supernatant was adjusted to 0.75 M NaCl, 2% β-mercaptoethanol. The samples were cooked in sealed tubes for 10 minutes, chilled on ice and clarified by centrifugation at 100,000 x g for 30 minutes. The supernatants were then adjusted to 2.5% perchloric acid and spun for 15 minutes at 13,000 x g. The precipitates were subjected to a second cycle of boil / acid precipitation and the pooled supernatants were dialyzed against 100 mM KH ₂ PO ₄ pH 6.9, 2 mM EDTA, 2 mM EGTA, 2 mM β-mercaptoethanol, 0.3 mM PMSF.

The recombinant tau was estimated to be at least 85% pure by SDS-PAGE stained with Coomassie blue and was used without further purification. The concentration of all the Taustandards were estimated by amino acid analyzes. To dephosphorylate tau, an aliquot was dialyzed in 20mM Tris-HCl pH 8.6, 2mM HgCl ₂ , 1mM DTT, 10μM ZnCl ₂ buffer. To the half of the sample was added 0.1 unit of alkaline phosphatase (Boehringer Mannheim) per μg of tau; the other half was similarly diluted with the buffer alone and the two samples were incubated for 5 hours at 37 ° C.

b. Monoclonal antibody against dew

monoclonal antibody were prepared according to a Modification of the method of Kohler and Milstein (G. Kohler and C. Milstein (1975) Nature 256: 495-497). Tau used in all Injections and screening assays were purified from SF9 cells, which were infected with the tau contained baculovirus construct. Six week old A / J mice were given with 100 μg injected on purified dew at two-week intervals. Dew was in complete Freund adjuvant involved for the first immunization and in incomplete Freund's adjuvant for all the rest Immunizations. The serum samples were taken three days after the third injection taken to evaluate the titer of these animals. The mouse with the highest Titer became intravenous with 100 μg on tau in 500 μl injected with PBS two weeks after receiving the third injection. Myeloma fusion occurred three days later using SP2 / 0 as the fusion partner. Antibodies 16G7 and SC11 were knocked out whereas antibodies 16B5 and 16C5 are derived from a subsequent fusion were isolated.

Supernatants from wells containing hybridoma cells were screened for their ability to precipitate ¹²⁵ I-labeled tau. Tau was radioiodinated using immobilized glycose oxidase and lactoperoxidase according to the manufacturer's instructions (Bio-Rad). Briefly, 10 μg of purified recombinant tau were radio labeled with 1 mCi of Na ¹²⁵ I to a specific activity of 20 μCi / μg protein. 16G7, 8C11, 16B5 and 16C5 were identified as the four monoclonal antibodies with the highest affinity specific for tau and were cloned by limiting the dilution. The isotypes from all four monoclonal antibodies specific for tau were determined to be gamma 1-kappa.

c. Tau ELISA

The anti-tau monoclonal antibody 16G7 was suspended at 5 μg / ml in TBS and coated at 100 μl / well in microtiter plates (Dynatec Microlite 2). The coating was carried out overnight at room temperature. The solution was then aspirated and the plates blocked with 0.25% casein (w / v) in phosphate buffered saline (PBS). The anti-tau antibody 16B5 was biotinylated with the N-hydroxysuccinimide ester of biotin according to the instructions of the manufacturer (Pierce). Samples of either 50 μl of CSF or calibrators (50 μl of 3-1000 pg / ml human tau) were mixed with 50 μl of the biotinylated anti-tau antibody (0.75 μg / ml in PBS-casein, 0.05% Tween 20 ) in the 16G7 coated wells and incubated at room temperature with constant shaking overnight. The solution was then aspirated and the plates were washed three times with TTBS. Streptavidin-alkaline phosphatase (Boehringer-Mannheim) was diluted 1: 1000 in PBS-casein, 0.05% Tween 20 and 100 μl added to each well. After incubation for 1 hour at room temperature, the liquid was aspirated and the wells were washed three times. The chemiluminescent reagent, disodium 3- (4-methoxyspiro {1,2-dioxetane-3,2 ¹ -tricyclo [3.3.1.1 ^3,7 ] tdecan} -4-yl) phenyl phosphate (AMPPD, Tropix) and an enhancer, Emerald Green (Tropix) were diluted 1: 1000 or 1: 100 in 1 M diethanolamine buffer containing 1 mM MgCl ₂ , 0.02% NaN ₃ , pH 10. 100 μl was added per well and the plates were read after 30 minutes in a Dynatech ML 1000 Chemiluminometer. The data presented here used human dew isolated from brain as the calibrator.

6. Statistical analysis

The Statistical analysis of the data was performed by one-way analysis Variance (ANOVA) using InStat version 1.21.

Results

Of the Comparison of the three patient groups (Table III) showed that they well matched in age and gender. The AD group showed an average MMSE of 17.5 ± 7.1, suggesting that mild to moderate cognitive damage was present. The neurological diseased control group consisted of a variety of diseases, inclusively vascular Dementia (4), forebrain dementia (7), depression (6), Parkinson's disease (3), Cortico-basal gangliene degeneration (2), cerebral ataxia (2), progressive supranuclear palsy (1), normal-pressure hydrocephalus (1), Grand mal seizure (1), Bell palsy (1), age-related memory damage (1), Dementia with extrapyramidal signs (1), amnetic syndrome (1), cerebral degeneration (1). The control group consisted of individuals, who did not show any neurological disorder and who were cognitively normal were (Table III).

The analysis of total CSF-Aβ levels did not reveal any significant differences among the different patient groups (Table III). The mean values ranged from 19.0 ng / ml in the AD group to 17.9 ng / ml in the NC group. There was significant overlap with no statistically significant differences among the groups (p> 0.05). However, analyzes of the Aβ (x-≥41) form of the peptide showed a mean reduction in the AD group, relative to both the ND and NC subjects (383 versus 543 and 632 pg / ml) significantly at the p <0.0001 level ( 4 ) was. The relatively small standard deviations (76 pg / ml) of the AD group were particularly noticeable. In contrast, some ND patients showed decreased Aβ (x-≥41) in their CSF. When a cutoff was set at 505 pg / ml, 15 of 37 ND patients and only four of 23 NC patients fell below this level. Alternatively was among the None of the individuals who had grade Aβ (x-≥41) greater than 505 pg / ml were found to have been diagnosed with AD, suggesting that the test is highly specific for the absence of disease. Aβ (x-≥41) was measured as described in the text. All measurements are average values of double value determinations, the variation was ≤ 10%. The samples were randomly evaluated on plates and the operator did not know the subject's diagnosis. The reference standards present on each microtiter plate were not significantly different between the plates.

Tau levels in the CSF samples of the same subject were also examined. The Taumessung was carried out twice. To ensure consistency, some samples from previous assays were trapped on subsequent plates and all samples were evaluated for at least one repeat measurement. Repeat measurements were within 15% of original values. A significant difference exists between the AD group and each control group (p <0.001). Tau derived from human brains was used as reference standard. AD patients had a mean of 407 pg / ml versus 168 and 212 pg / ml in neurological and normal controls, respectively ( 5 ). This difference between the AD group and the other groups is significant at p <0.001. Using a cut-off of 312 pg / ml, individuals with levels above this level showed a very high probability of Alzheimer's disease (22/24 = 92%). Only one NC and one ND subject was registered above this cutoff. Separate analysis of mean CSF Aβ, Aβ (x-≥41) or trough levels obtained from each center showed no differences between the centers that were statistically significant for any of the disease categories, as demonstrated by a one-way analysis variance has been. Of particular interest was the simultaneous analysis of Aβ (x-≥41) and Taumessungen in the same CSF samples ( 6 ). 6 is then divided in Figure using the cutoffs for Aβ (x-≥41) and Tau as previously described. The presence of both elevated tau and decreased Aβ (x-≥41) (lower right quadrant) was of highest prognostic significance for AD (22/23 = 96%). On the other hand, high Aβ (x-≥41) and low tau (lower left quadrant) were completely represented by control patients ( 6 ). More than half (48.7%) of all individuals in this study fell into one of these two quadrants.

The remaining patients showed low Aβ (x-≥41) and low tau levels (lower left quadrant).

Even though the foregoing invention in detail for the purpose of clarity and of understanding has been described, it will be obvious that certain modifications within the scope of the attached claims can be practiced.

SEQUENCE LISTING

Claims (19)

A method useful as a part of a diagnostic Procedure for Alzheimers disease on a patient, said method comprising: measure up the amount of one or more soluble Aβ (x-≥41) in a sample of body fluid a patient; Comparison of the measured quantity with a predetermined one Indicator value, from said one or more soluble Aβ (x-≥41), in which optionally, the predetermined indicator value from the same patient an earlier time was measured and the procedure ensures monitoring; assess the condition of the patient based on the difference between the measured amount and the predetermined indicator value; and wherein a measured amount above the indicator value is a negative one Indication in the diagnosis of Alzheimers disease and a measured value of or below the indicator value is a positive one Indication in the diagnosis of Alzheimers disease. A method as claimed in claim 1, wherein the patient sample is a cerebrospinal fluid and the indicator value between about 0.45 ng / ml and about 0.7 ng / ml, preferably about at 0.5 ng / ml and optionally wherein the Aβ (x-≥ 41) was measured, at least which contains Aβ amino acids 33-41. A method as claimed in claim 1, wherein the patient sample is plasma. A method as claimed in claims 1 to 3, wherein the amount of soluble Aβ (x-≥ 41) is going through Exposing the patient sample to first antibodies or a fragment thereof, specific for the binding region on Aβ or Aβ fragment spanning amino acid residues 13 to 26, and Detecting the bond between the first antibody or fragment thereof and the soluble one Aβ (x-≥ 41) Exposing the patient sample to a second antibody or a fragment thereof, specific for Aβ (x-≥41), in which preferably (a) the second antibody or fragment thereof Recognizes epitope of Aβ, the amino acid residues 33-42 has, and / or (b) the first antibody or fragment thereof Recognizes epitope on Aβ, that the amino acid residues 13-26 having. A method as claimed in claim 4, wherein the binding of the first antibody or fragment thereof and the soluble Aβ (x-≥41) is detected by separating bound complexes of the first antibody or fragment thereof and Aβs or Aβ fragments, wherein these separately bound complexes are exposed to a labeled second antibody or fragment thereof, specific for Aβ (x-≥41), and by detecting the presence of the label on the bound complexes. A method as claimed in claim 1, wherein the amount of soluble Amyloid-β Peptide (x-≥41) (Aβ (x-≥41)) in the Patient sample, optionally the cerebrospinal fluid, is measured by capture of the soluble Aβ (x-≥ 41) from the Sample using a first antibody or fragment thereof, specific for a binding region spanning Aβ amino acid residues 13 to 26; and Detecting the capture of soluble Aβ (x-≥ 41) under Use of a labeled second antibody or fragment thereof specific for Aβ (x-≥ 41). A method as claimed in claim 6, wherein the soluble Aβ (x-≥ 41) on one solid phase is captured and the capture is detected by Exposure of the solid phase to a second antibody or Fragment of it and by subsequently detecting the presence the label on the solid phase. A method for screening a compound for its ability to determine the amount of Aβ (x-≥ 41) in the To change CSF; the method comprising: Measuring a first set of one or more soluble Aβ (x-≥41) in the CSF of a non-human animal used as a model for Alzheimer's Disease is used; Administering the compound to the non-human animal; Measuring a second quantity of said one or more soluble Aβ (x-≥41) in the CSF of the non-human animal; and Comparison of the first Quantity with the second quantity, with the difference indicating whether the Compound the amount of soluble Aβ (x-≥41) in the CSF increased, decreased or unchanged leaves, wherein a compound which increases the amount of Aβ (x-> 41) in the CSF is useful in the treatment Alzheimer's disease and a compound that uses the amount of Aβ (x-> 41) can aggravate Alzheimer's disease or can accelerate. A method useful as a part of a diagnostic Procedure for Alzheimers Disease in a patient, said method includes: Measuring the amount of one or more soluble Aβ (x-≥ 41) in one Sample of a body fluid in a patient; Comparison of the measured amount of soluble Aβ (x-≥ 41) with one predetermined amount of indicative soluble Aβ (x-≥41), Measuring the amount of Dew of the patient sample; Comparison of the measured amount of Tau with a predetermined indicator value of Tau; and assess patient status based on the difference between the measured Quantities and the predetermined indicator values of Aβ (x-≥41) and Tau, wherein a measured amount of or below the Aβ (x-> 41) and from or above the tau indicator value is a provides a positive indication in the diagnosis of Alzheimer's disease, and wherein the measured amount is above the Aβ (x-> 41) indicator value and below the Tau indicator value is a negative indication in the diagnosis of Alzheimers disease provides. A method as claimed in claim 9, wherein the patient sample is a cerebrospinal fluid, the indicator value for Aβ (x-≥ 41) between approximately 0.45 ng / ml and about 0.7 ng / ml, preferably about at 0.5 ng / ml and the indicator value for tau is between about 0.25 ng / ml and about 0.4 ng / ml, preferably about at 0.3 ng / ml, optionally with the measured Aβ (x-≥ 41) at least which contains Aβ amino acids 33-41. A method as claimed in claim 9, wherein the patient sample is plasma. A method as claimed in claims 9 and 11, wherein the amount of soluble Aβ (x-≥ 41) is going through Exposing the patient sample to a first antibody or a fragment thereof specific for the binding region on Aβ or Aβ fragment spanning amino acid residues 13 to 26 is, and Detecting the bond between the first antibody or fragment thereof and the soluble one Aβ (x-≥ 41) Exposing the patient sample to a second antibody or a fragment thereof specific for Aβ (x-≥ 41). A method as claimed in claim 12, wherein a. the second antibody or fragment thereof is an antibody recognizing an epitope of Aβ having amino acid residues 33-42, and / or b. the first antibody or fragment thereof is an antibody which recognizes an epitope of Aβ having amino acid residues 13-26. A method as claimed in claim 12 or 13, the binding of the first antibody or fragments thereof and the soluble one Aβ (x-≥ 41) detected is going through Separate bound complexes of the first antibody or Fragments thereof and Aβ or Aβ fragments, these separately bound complexes being labeled a second antibody or fragment thereof, specific for Aβ (x-≥41), and detect the presence of the label on the bound complexes. A method for screening a compound to their ability to determine the amount of both Aβ (x-≥41) and tau in the CSF to change, the method comprising: Measuring a first set of one or more soluble Aβ (x-≥41) in the CSF of a non-human animal used as a model for Alzheimer's Illness; Measuring a first amount of tau in the CSF of a non-human animal; Administer the compound to non-human animal; Measuring a second quantity of said one or more soluble Aβ (x-≥41) in the CSF of the non-human animal; Measuring a second quantity of tau in the CSF of the non-human animal; and to compare the first quantity with the second quantity, the difference indicates whether the compound increases, decreases or leaves the amount of soluble Aβ (x-≥41) and whether it increases, decreases or leaves unchanged the amount of tau in the CSF, where a compound that increases the amount of Aβ (x-> 41) and the amount of tau diminished in a CSF, useful in the treatment of Alzheimers Disease, and a compound that reduces the amount of Aβ (x-> 41) and the amount of dew increases, Alzheimers Illness can worsen or accelerate. A method as in claim 8 or claim 15 wherein the non-human animal is a rodent, preferably a mouse. A kit comprising an antibody or fragment thereof which Aβ (x-≥ 41) binds, however, does not bind Aβ (≤40) and an antibody or fragment thereof which binds tau, optionally further comprising an antibody or fragment thereof, which Aβ or binds a fragment of Aβ, however, does not bind other fragments of APP. A kit as claimed in claim 17, wherein the antibody or fragment thereof which binds Aβ (x-≥41), but not at A (≤ 40) binds to an epitope containing amino acids beyond number 40 in Aβ binds, and the antibody or fragment thereof, which Aβ or binds a fragment of Aβ, however, does not bind other fragments of APP to a binding region spanning Aβ Amino acid residues 13 binds to 26 A kit as claimed in claim 18, comprising (A) an unlabeled antibody or fragment thereof, which is a binding region of Aβ spanning amino acid residues 13 to 26 binds; (b) a detectably labeled antibody or Fragment thereof, which binds an epitope, the amino acids by number 40 going into Aβ contains; (C) an unlabeled antibody or fragment thereof which binds to tau; and (d) one detectable labeled antibody or fragment thereof which binds to tau; are preferred the antibodies monoclonal antibodies or fragments thereof.